1. Technical Field
The present invention relates to methods of altering cells to enhance production of proteins they have been raised to express. Particularly, this invention addresses the use of Random Homozygous Gene Perturbation to enhance antibody expression of an antibody-expressing host, by targeted insertion of DNA to either depress endogenous expression of a host protein, or enhance expression of a poorly expressed host protein, the change in expression being related to an increase in expression of the antibody expressed by the host cell.
2. Background of the Technology
Antibodies, particularly monoclonal antibodies, have become important biologic products both in mankind's arsenal against disease, and in research and development. While not the “magic bullet” once envisioned, more than a score of monoclonal antibodies, sometimes referred to as mAb, have been approved for therapeutic use. Just a few of these include the Trastuzumab antibody, the active agent in Herceptin® approved for the treatment of some breast cancers, Palivizumab, the mAb of Synagis® approved for the prevention/treatment of RSV, and Bevacizumab, a mAb present in Avastin®, approved for the treatment of colorectal cancer, and indicated to be effective in treating other conditions. Many more are known.
By contrast, there are literally thousands of antibodies, mAb and polyclonal, employed as workhorses in laboratories and research facilities around the world. Antibodies are useful as diagnostics, as agents to bind and isolate target molecules, to differentiate cells for testing, and other uses that take advantage of the specific binding properties of IgG to select out a single antigen, typically a biological molecule, bound or unbound, that may be of interest. Antibody production is fundamental business.
Methods of making antibodies are well established, although refinements are added constantly. The basic information was set forth as early as 1975, Kohler & Milstein, Nature, 256: 495-497 (1975). To prepare monoclonal antibodies, a host, typically a rabbit or the like, is injected with the antigen against which a mAb is sought. Following immunization, the spleen, and possibly lymph nodes, of the host are removed and separated into single cells. These cells are then exposed to the target antigen. Cells that express the desired mAb on their surface will bind to the immobilized antigen. These cells are cultured and grown, and fused with myeloma cells or other immortal cells to form hybridoma, which can be cultured to recover the expressed antibody.
Most antibodies, and virtually all therapeutic antibodies, need to be modified to avoid inducing a rejection reaction in a patient. The DNA encoding the antibody expressed by the hybridoma is isolated, and can be modified by the insertion or removal of bases, altered glycosylation profiles, and manipulation of framework regions and complementary determining regions, which affect the affinity and avidity with which the antibody binds to its target antigen. The resulting antibodies are humanized or “human” or otherwise modified (chimeric antibodies and veneered antibodies are common in the art). The state of the art as of about 1995 is reflected in U.S. Pat. No. 6,054,561, the relevant disclosure of which is incorporated herein by reference.
Once prepared and isolated, the DNA encoding the antibody may be transferred to a preferred mammalian cell line for expression in “production” or commercial amounts. It has long been recognized that Chinese Hamster Ovary cells (CHO cells) make excellent expression vehicles for recombinant or non-endogenous DNA. See U.S. Pat. No. 4,816,567. There has been developed a series of DHFR deficient CHO cell strains, which permit the amplification of inserted DNA encoding specific proteins or DNA sequences, as set forth in U.S. Pat. No. 5,981,214. This latter patent describes the use of homologous recombination to target a specific gene or expression region of a cell—in the case in question, to induce expression of a heterologous gene. Other suitable cell lines include 293HEK cells, HeLa cells, COS cells, NIH3T3 cells, Jurkat Cells., NSØ cells and HUVEC cells. Other mammalian cell lines suitable for the expression of recombinant proteins have been identified in the literature, and are equally suitable for use in the invention of this application.
Once stabilized, current methods to increase production of the valuable antibodies tend to focus on increases the total productivity, that is, high volumetric productivity, so that a given amount of cells produces a given amount of antibodies. These methods tend to focus on improving the methods and environments used to cultivate the cells, to enhance total antibody production. In general, antibody production of greater than about 1 g/L is required for an industrially competitive process. Individual CHO cells are typically expressing in the range of 10-15 pg/cell/day.
Homologous recombination has been used in many contexts since about 1985. It was originally employed as a “knock-out” tool, allowing the suppression of an expressed gene, to study the response of the modified cell. Subsequent procedures were developed to allow the silencing of target genes. The use of anti-sense knock out constructs using a random homozygous knock out method (RHKO) is described, e.g., in Li et al, Cell 85: 319-329 (196). In U.S. Patent Publication 20060240021 (U.S. patent application Ser. No. 10/524,426 filed Aug. 18, 2003) the use of RHKO techniques is disclosed to identify the genes involved in rapamycin resistance. The entirety of that disclosure is incorporated herein by reference. The ability to insert a construct into one allele, identify the cells where that allele has been successfully modified by quick throughput searching, such as for example by FACS (fluorescence activated cell sorter) and similar methods makes this a superior technique for selective identification and modification of a cell's genome. U.S. Pat. No. 6,835,816, incorporated by reference herein discloses the use of this technique in conjunction with genes reflecting tumor susceptibility, including TSG101 genes.
Accordingly, it remains a goal of the industry to find a way to increase the expression of antibodies, particularly recombinantly prepared antibodies, from expression hosts like CHO cells, 293HEK cells, HeLa cells, COS cells, NIH3T3 cells, Jurkat Cells, NSØ cells and HUVEC cells. and others, in a stable and reproducible fashion, using available techniques to modify the genome of the cell.